The concept behind fluorescence microscopy is as follows. Fluorescent dye molecules can be attached to specific parts of the biological sample of interest. When excited by a suitable wavelength of light these markers fluoresce so that only those parts are seen in the microscope. More than one type of fluorescent dye can be used in the sample to attach to different features. By changing the wavelength of the excitation light, different types of dye can be forced to fluoresce thus enabling different structures within the sample to be distinguished. The dyes can be chosen to fluoresce at different wavelengths. Thus if the sample is excited by a spectrum of light the colour (or wavelength) of the fluorescence will give information as to the structure. In laser scanning microscopy, for example, a laser will scan the sample and the fluorescence observed by a suitable detector. A schematic representation of such a system is shown in FIG. 1.
The laser scanning microscopy device of FIG. 1 comprises a laser 10 arranged to illuminate a sample 12 via a dichroic mirror and two scanning mirrors 16, 18. The scanning mirrors cause the laser beam to scan the surface of the sample 12. Imaging optics 20 are arranged so that the scanning spot of light in the sample is imaged onto a pin hole 22. This arrangement is referred to as a confocal arrangement and ensures that only fluorescence from a particular spot being scanned reaches a detector 24. Any light from a depth within the sample that is not at the focus of the optics will not be focussed exactly on the pin hole (it will form an Airy ring larger than the pin hole) and so will not pass to the detector 24. The confocal arrangement thus allows different portions of a sample to be sampled over time (as the beam scans) and so this is a form of time resolved spectroscopy.
Multiple lasers 10 at different wavelengths can also be used to scan the sample and the spectrum of the fluorescence can be sampled. A picture of the distribution of the fluorescent markers, and thus the structure can thus be constructed. This is illustrated in FIG. 2. Like components are given the same reference numerals as FIG. 1 and the description will not be repeated here. A prism or diffraction grating 26 splits the spectrum of wavelengths from the sample across a plurality of sensors 28. This allows the spectral response of the sample at different wavelengths to be simultaneously sampled.
The way the fluorescent spectrum is split between the detectors is determined by the prism/diffraction-grating configuration. This is chosen to separate out the markers of interest or to sample the spectrum of light from one marker for example. The current state of the art utilises a small number of discrete detectors (e.g. 4), each sampling a range of wavelengths. The detectors currently employed are typically photomultiplier tubes or avalanche photo diode devices. There is currently a need to take a more complete sample of the emitted spectrum in order, for example, to simplify the optics, and to present more information to the operator so that a flexible use of optical filters can be applied in software once the data has been acquired. Ideally a detector that can sample the spectrum simultaneously at wavelengths between 300 and 900 nm and a resolution of, say 10 nm would be useful. Typical requirements are for the sample to be scanned to produce an image with a resolution of 512×512 pixels at a rate in the order of 5 images per second. This implies that a full spectra will need to be acquired at a rate of about 1,300,000 spectra per second; that is one spectra needs to be acquired every 0.8 micro seconds. In practice the required rate of spectra acquisition will be higher than this as a fraction of the scanning time will be required for the change in the direction of the scan, for example.
For conventional non-scanning digital spectral detection, for example those used within spectrometer systems, linear or area array charge coupled (CCDs) are employed to image the spectrum dispersed by a diffraction grating. In general these systems have poor temporal resolution. The spectrum is distributed across the imaging area and the resultant electrons generated by the incident photons are collected within the pixels during the integration time. The signal is then read out of the devices via a readout register and on-chip charge to voltage conversion amplifiers. Various modes of operation can be employed, for example full vertical binning can be used. A CCD architecture can be used that enables the so-called “kinetic mode” of operation. This architecture is illustrated in FIG. 3. Here an area array 36 is completely masked off from the incident light except for a small number of lines 32 (typically one) at the top of the device. Spectra, once obtained, can be clocked rapidly into the storage region 36 of the device and then to an output amplifier 30. Thus spectra with good temporal resolution can be gathered. However, once the storage region is filled it has to be readout serially via the readout register. This is a much slower process than the transfer into the store and would not meet the timing requirement of the laser scanning system. More importantly, a system based on a conventional CCD or CMOS device will not have the required sensitivity. This is determined by the need to reduce the illumination incident on the sample to the minimum to avoid bleaching effects and sample damage.